Summary of Work: The complex and poorly understood process of lineage progression from neural stem cells to neuronal and glial phenotypes has come under increasing study using a variety of in vitro strategies. An important issue to be resolved is how neural stem cells are regulated to either self-renew or differentiate. In vivo these cells line the ventricles of the developing central nervous system (CNS) with variable numbers radiating processes to the pial surface (radial glial form of neural stem cell). The cells integrate signals derived from both carrier proteins in the cerebrospinal fluid filling the ventricles and other cells in the neuroepithelium lining the ventricles. Signals conveyed by carrier proteins and via cell-cell interactions, which could serve to regulate cell lineage progression, have not been eluciated. We have developed a novel strategy to isolate identified subpopulations of neural stem cells and differentiaing progenitors from the CNS during neurogenesis and gliogenesis. The strategy involves labeling of live cells with reagents identifying surface gangliosides, which are conserved throughout vertebrate evolution, and epitopes characteristic of cells undergoing apoptosis in conjunction with fluorescence-activated cell sorting (FACS). This FACS strategy permits prospective cellular and molecular studies of neural stem cells for the first time. During FY 2003 we focused primarily on elucidating the role of basic fibroblast growth factor (bFGF) in neural stem cell biology. A novel multi-epitope staining strategy was developed in conjunction with FACS analysis to reveal that the majority of vital neural stem cells (NSCs) in proliferation in vivo express one of the primary receptors for bFGF (fibroblast growth factor receptor 1, FGFR-1). Another novel strategy was used to record Ca2+ levels of NSCs with FACS. The same percentage of NSCs as expressed FGFR-1 responded to bFGF with a rise in Ca2+ levels. These effects recorded ex vivo involve both Ca2+ entry and Ca2+ release components, which are activated downstream of FGFR-1 autophosphorylation at tyrosine sites. NSCs self-renewing in vitro as undifferentiated precursors exhibited similar Ca2+ responses to bFGF. These results implicate bFGF/FGFR-1 mediated Ca2+ signaling in vitro in maintaining NSCs in an undifferentiated, proliferative state. By extension, the results suggest that similar levels of bFGF- regulated Ca2+ levels promote NSC self-renewal in vivo. NSC self-renewal without overt differentiation could be suppressed and NSC progression along a neuronal lineage induced by decreasing the concentration of bFGF 10-100 fold. Similar effects occurred when the tyrosine kinase activity resulting from bFGF/FGFR-1 signaling was reduced using a selective inhibitor. At high doses of inhibitor NSCs died rather than differentiated or divided. Thus, the level of tyrosine kinase activity associated with bFGF/FGFR-1 signaling determines both the regulation of intracellular Ca2+ levels and the fate of NSCs in vitro. Injection of the tyrosine kinase inhibitor in vivo noticeably reduced cortical growth in select regions. FACS analysis showed that compared to normal development there was a marked decrease in NSC proliferation and self-renewal as well as an increase in cell death. These results implicate bFGF/FGFR-1 tyrosine kinase activation in vivo in determining NSC fate. Since the inhibitor blocks tyrosine kinase activation of all four FGFRs, it is not clear how many mediate the effects of bFGF. Pharmacological experiments on the dowstream pathways linking bFGF/FGFR-1 signaling to regulation of intracellular Ca2+ levels revealed at least four different enzymatic circuits to be activated. The products of the enzymatic activities target Ca2+ release from intracellular stores as well as Ca2+ entry, most likely via cation-selective channels whose activity is independent of cellular potential. At least four candidate channels have been identified. How different levels of FGFR tyrosine activation regulate Ca2+ levels and how these are related to NSC fate remain challenges for future study. Preliminary experiments show that classical neurotransmitters can also regulate Ca2+ levels as NSCs progress along different lineages. The role of transmitter signaling in determining NSC fate will also be pursued.